RESEARCH ARTICLE

Respiratory Syncytial Virus Fusion Protein Promotes TLR-4–Dependent Neutrophil Extracellular Trap Formation by Human Neutrophils Giselle A. Funchal1,2,4, Natália Jaeger2,4, Rafael S. Czepielewski2,4, Mileni S. Machado1,4, Stéfanie P. Muraro1,4, Renato T. Stein3,4, Cristina B. C. Bonorino2,4, Bárbara N. Porto1,3,4* 1 Clinical and Experimental Immunology Laboratory, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil, 2 Cellular and Molecular Immunology Laboratory, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil, 3 Infant Center, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil, 4 Institute of Biomedical Research, Pontifícia Universidade Católica do Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil * [email protected]

OPEN ACCESS Citation: Funchal GA, Jaeger N, Czepielewski RS, Machado MS, Muraro SP, Stein RT, et al. (2015) Respiratory Syncytial Virus Fusion Protein Promotes TLR-4–Dependent Neutrophil Extracellular Trap Formation by Human Neutrophils. PLoS ONE 10(4): e0124082. doi:10.1371/journal.pone.0124082 Academic Editor: Ralph Tripp, University of Georgia, UNITED STATES Received: September 9, 2014 Accepted: March 6, 2015 Published: April 9, 2015 Copyright: © 2015 Funchal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) grant nr. 472406/2010-8, and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) grant nr. 11/1904-1. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Abstract Acute viral bronchiolitis by Respiratory Syncytial Virus (RSV) is the most common respiratory illness in children in the first year of life. RSV bronchiolitis generates large numbers of hospitalizations and an important burden to health systems. Neutrophils and their products are present in the airways of RSV-infected patients who developed increased lung disease. Neutrophil Extracellular Traps (NETs) are formed by the release of granular and nuclear contents of neutrophils in the extracellular space in response to different stimuli and recent studies have proposed a role for NETs in viral infections. In this study, we show that RSV particles and RSV Fusion protein were both capable of inducing NET formation by human neutrophils. Moreover, we analyzed the mechanisms involved in RSV Fusion protein-induced NET formation. RSV F protein was able to induce NET release in a concentration-dependent fashion with both neutrophil elastase and myeloperoxidase expressed on DNA fibers and F proteininduced NETs was dismantled by DNase treatment, confirming that their backbone is chromatin. This viral protein caused the release of extracellular DNA dependent on TLR-4 activation, NADPH Oxidase-derived ROS production and ERK and p38 MAPK phosphorylation. Together, these results demonstrate a coordinated signaling pathway activated by F protein that led to NET production. The massive production of NETs in RSV infection could aggravate the inflammatory symptoms of the infection in young children and babies. We propose that targeting the binding of TLR-4 by F protein could potentially lead to novel therapeutic approaches to help control RSV-induced inflammatory consequences and pathology of viral bronchiolitis.

PLOS ONE | DOI:10.1371/journal.pone.0124082 April 9, 2015

1 / 14

RSV F Protein Induces NET Formation

Competing Interests: Co-author Cristina Bonorino is a PLOS ONE Editorial Board member. This does not alter the authors' adherence to PLOS ONE Editorial policies and criteria.

Introduction Respiratory Syncytial Virus (RSV)-induced acute bronchiolitis is the most prevalent respiratory disease in children under age 2 years, and its seasonal epidemics are associated with a significant number of hospital admissions, with a huge burden to communities worldwide [1]. Almost 70% of all children are infected with RSV during the first year of life, and by age 3, practically all children will have experienced at least one infection with this virus [2, 3]. RSV is a single stranded RNA virus, whose genome encodes up to 11 proteins [4]. The Fusion (F) protein, present at the virion surface, mediates fusion of the viral envelope with the target cell membrane during virus entry [5]. Only membrane-bound F protein is indispensable for virus replication in vitro and in vivo [6], and this protein is the primary target for both antiviral drug and vaccine developments [7, 8]. It has been demonstrated that RSV F protein activates pattern recognition receptors TLR-4 and CD14, inducing pro-inflammatory cytokine secretion [9]. In addition, it has been recently shown that RSV F protein directly interacts with the MD-2–TLR-4 complex, thus activating the transcription factor NF-κB [10]. These studies highlight the importance of specific signaling pathways activated by F protein to stimulate inflammation. One of the characteristic features of RSV infection is the large amounts of neutrophils in the lower airways once infection is established [11]. It is also well recognized that neutrophils and their products are present in the airways of patients and animal models with RSV bronchiolitis [11–13], and also in virus-induced asthma [14, 15]. This body of evidence suggests that neutrophils play an important role in the pathogenesis observed in the airways of affected children [16,17]. Aside from the traditional mechanisms of phagocytosis, generation of reactive oxygen species (ROS), and degranulation, neutrophils can also produce neutrophil extracellular traps (NETs), an important strategy to immobilize and kill pathogens [18]. NETs are formed by decondensed chromatin fibers decorated with antimicrobial proteins, such as neutrophil elastase and myeloperoxidase [18]. NET-inducing stimuli include cell surface components of bacteria, such as LPS, whole bacteria, fungi, protozoan parasites, cytokines, and activated platelets, among others [18–22]. More recently, studies have demonstrated that viruses are also capable of inducing NET formation. In vitro, the production of NETs is modulated in neutrophils isolated from cats infected with feline immunodeficiency virus [23]. NETs activated after infection by Human Immunodeficiency Virus (HIV-1) are crucial for the elimination of virus [24]. NET release in the liver vasculature also protects host cells from poxvirus infection [25]. However, an excessive production of NETs contributes to the pathology of respiratory viral infections. NET formation is potently induced in lungs of mice infected with Influenza A virus, in areas of alveolar destruction [26], suggesting a putative role for NETs in lung damage. We show that RSV virion was able to induce NET formation by human neutrophils and RSV F protein stimulated NET formation dependent on TLR-4 receptor activation. Moreover, F protein-induced NETs were decorated with neutrophil elastase and myeloperoxidase, granule proteins that can damage tissues. F protein potently induced NADPH Oxidase-derived ROS production and this was crucial for NET generation. Also, F protein induced NET production in an ERK and p38 MAPK phosphorylation-dependent manner. Together, these results provide compelling evidence to support a signaling mechanism activated by RSV F protein to induce NET formation. The massive production of NETs in the airways of children infected with RSV may worsen lung pathology and impair lung function.

PLOS ONE | DOI:10.1371/journal.pone.0124082 April 9, 2015

2 / 14

RSV F Protein Induces NET Formation

Materials and Methods Reagents RSV A2 strain was provided by Dr. Fernando Polack (Vanderbilt University School of Medicine, USA). Human recombinant RSV Fusion protein was purchased from Sino Biological Inc. According to the manufacturer, the glycosylated protein purity is >95% and endotoxin level is

Respiratory syncytial virus fusion protein promotes TLR-4-dependent neutrophil extracellular trap formation by human neutrophils.

Acute viral bronchiolitis by Respiratory Syncytial Virus (RSV) is the most common respiratory illness in children in the first year of life. RSV bronc...
592KB Sizes 1 Downloads 6 Views